Akiko Uehara

Neutrophil Proteinase 3-Mediated Induction of Bioactive IL-18 Secretion by Human Oral Epithelial Cells

IL-18, a potent IFN-γ-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-γ-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-γ-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-γ-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.

Muramyldipeptide and diaminopimelic acid‐containing desmuramylpeptides in combination with chemically synthesized Toll‐like receptor agonists synergistically induced production of interleukin‐8 in a NOD2‐ and NOD1‐dependent manner, respectively, in human monocytic cells in culture

Two types of synthetic peptidoglycan fragments, diaminopimelic acid (DAP)‐containing desmuramylpeptides (DMP) and muramyldipeptide (MDP), induced secretion of interleukin (IL)‐8 in a dose‐dependent manner in human monocytic THP‐1 cells, although high concentrations of compounds are required as compared with chemically synthesized Toll‐like receptor (TLR) agonists mimicking bacterial components: TLR2 agonistic lipopeptide (Pam3CSSNA), TLR4 agonistic lipid A (LA‐15‐PP) and TLR9 agonistic bacterial CpG DNA. We found marked synergistic IL‐8 secretion induced by MDP or DAP‐containing DMP in combination with synthetic TLR agonists in THP‐1 cells. Suppression of the mRNA expression of nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the synergistic IL‐8 secretion induced by DMP and MDP with these TLR agonists respectively. In accordance with the above results, enhanced IL‐8 mRNA expression and the activation of nuclear factor (NF)‐κB induced by MDP or DMP in combination with synthetic TLR agonists were markedly suppressed in NOD2‐ and NOD1‐silenced cells respectively. These findings indicated that NOD2 and NOD1 are specifically responsible for the synergistic effects of MDP and DMP with TLR agonists, and suggested that in host innate immune responses to invading bacteria, combinatory dual signalling through extracellular TLRs and intracellular NODs might lead to the synergistic activation of host cells.

Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2

1. Figs. 1B and 2A: Total RNAwas extracted from different cells, and cDNAwas prepared and analyzed for the expression of SLPI and PARs and GAPDH by RT-PCR. However, the patterns of GAPDH in these figures and those in the figures of The Journal of Immunology, 2002, 169: 4594–4603 and in Fig. 2A of Clinical and Diagnostic Laboratory Immunology, 2003, 10: 286–292 are the same. 2. Fig. 2B: Two panels of the expression of PAR-2 with HLE and Cat G are the same.

Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of β-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Functional TLRs and NODs in Human Gingival Fibroblasts

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-κB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.

Chemically synthesized pathogen‐associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll‐like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti‐bacterial factors. In humans, four kinds of PGRPs (PGRP‐L, ‐Iα, ‐Iβ and ‐S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized  pathogen‐associated  molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli‐type tryacyl lipopeptide (Pam3CSSNA), E. coli‐type lipid A (LA‐15‐PP), diaminopimelic acid containing desmuramyl peptide (γ‐ d‐glutamyl‐meso‐DAP; iE‐DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP‐Iα and ‐Iβ, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll‐like receptor (TLR)2, TLR4, nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA‐15‐PP, iE‐DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)‐κB in the epithelial cells, and suppression of NF‐κB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Priming of human oral epithelial cells by interferon-γ to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans

An earlier study reported that human gingival epithelial cells in primary culture and oral epithelial cell lines KB and HSC-2 cells were devoid of membrane CD14 (mCD14) and did not show enhanced production of interleukin (IL)-8 or granulocyte macrophage-colony stimulating factor (GM-CSF) upon stimulation with bacterial cell-surface components such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN) and synthetic muramyldipeptide (MDP) even in the presence of serum. The present study demonstrated that after treatment with interferon (IFN)-gamma for 3 days, these cells secreted IL-8 and GM-CSF in response to the bacterial components. Treatment with IFN-gamma enhanced Toll-like receptor (TLR) 2, TLR4, MD-2 and MyD88 mRNA expression as determined by reverse transcriptase PCR. Anti-TLR2 and anti-TLR4 monoclonal antibodies (MAbs) inhibited the IL-8 production induced by PGN and LTA as well as LPS, respectively, in IFN-gamma-primed oral epithelial cells, whereas neither MAb inhibited IL-8 production induced by MDP. These findings suggested that IFN-gamma primed oral epithelial cells to produce cytokines upon stimulation with various bacterial components by up-regulation of the TLR system.

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

1. Fig. 1E: HSC-2, an oral epithelial cell line, is different from KB cells, but the patterns of their band staining for IL-8, MCP-1, and GAPDH cDNA are the same. 2. Fig. 2D: Bands for MCP-1 and GAPDH cDNA in Fig. 1E and ICAM-1 and GAPDH cDNA in this figure are the same. 3. Fig. 4A: Total RNAwas extracted from KB (lane 2), HSC-2 (lane 3), and PBMCs (lane 4), and cDNAwas prepared and analyzed for the expression of PAR1-4 and GAPDH by RT-PCR. However, three bands of PAR3 and a band of PAR4 are the same. Furthermore, the patterns of GAPDH in this article and those in Fig. 1B and 2A of The Journal of Immunology, 2003, 170: 5690–5696 and in Fig. 2A of Clinical and Diagnostic Laboratory Immunology, 2003, 10: 286–292 are the same. 4. Fig. 4B and 4D: Three panels of the expression of PAR1 with PR3 and PR3 + cytochalasin B (Cyto B) and PAR3 with PR3 + Cyto B are the same. Two panels of the expression of PAR2 with PR3 + cycloheximide (CHX) in Fig. 4B and with trypsin in Fig. 4D are the same.

Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll‐like receptor‐2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o‐. Treatment of the cells with 5‐azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non‐CF 16HBE14o‐ cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o‐ cells, implying that decreased methylation of the TLR2 promoter is responsible for CF‐related up‐regulation of TLR2. Finally, stable expression of WT‐CFTR in CFBE41o‐ cells (CFBE41o‐/WT‐CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up‐regulation. Consistent with reduced expression of TLR2 in CFBE41o‐/WT‐CFTR cells, CpG methylation was increased in CFBE41o‐/WT‐CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up‐regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

Meso-diaminopimelic acid and meso-lanthionine, amino acids specific to bacterial peptidoglycans, activate human epithelial cells through NOD1.

Peptidoglycans (PGNs) are ubiquitous constituents of bacterial cell walls and exhibit various immunobiological activities. Two types of minimum essential PGN structures for immunobiological activities were chemically synthesized and designated as muramyldipeptide; N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) and γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), which are common constituents of both Gram-positive and Gram-negative bacteria, as well as most Gram-negative and some Gram-positive bacteria, respectively. Recently, intracellular receptors for MDP and iE-DAP have been demonstrated to be nucleotide-binding oligomerization domain (NOD)1 and NOD2, respectively. In this study, we demonstrated that chemically synthesized meso-DAP itself activated human epithelial cells from various tissues, through NOD1 to generate antibacterial factors, PGN recognition proteins and β-defensin 2, and cytokines in specified cases, although the activities of meso-DAP were generally weaker than those of known NOD agonists. However, stereoisomers of meso-DAP, ll-DAP, and dd-DAP were only slightly activated or remained inactive, respectively. Synthetic meso-lanthionine, which is another diamino-type amino acid specific to PGN of the specified Gram-negative bacteria, was also recognized by NOD1. In human monocytic cells, in the presence of cytochalasin D meso-DAP induced slightly but significantly increased production of cytokines, although the cells did not respond to meso-DAP in the absent of cytochalasin D. Our findings suggest that NOD1 is a special sentinel molecule, especially in the epithelial barrier, allowing the intracellular detection of bacteria through recognizing meso-DAP or comparable moiety of PGN from specified bacteria in cooperation with NOD2, thereby playing a key role in innate immunity.